Combined deletion of Hfe and transferrin receptor 2 in mice leads to marked dysregulation of hepcidin and iron overload

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2; Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin. Conclusion: These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8. (HEPATOLOGY 2009.)

Iron is an essential element, required for numerous metabolic processes. A deficiency of iron can lead to anemia, whereas an excess can be toxic and lead to tissue damage and disease, as in hereditary hemochromatosis (HH). Iron homeostasis is tightly controlled through the regulation of duodenal iron absorption and iron release from macrophages and other cell types. The liver plays a central role in the regulation of body iron metabolism and many of the proteins mutated in the various forms of HH are expressed at high levels in the liver. Hepcidin, a small peptide hormone expressed predominantly in the liver, is synthesized and secreted in response to iron loading or inflammation and lowers serum iron levels by binding the iron exporter ferroportin, causing its internalization and degradation.1

Most forms of HH result from mutations in the genes involved in regulating hepcidin expression and manifest in inappropriately low levels of hepcidin in relation to iron stores. Mutations in hepcidin (HAMP) and hemojuvelin (HJV) lead to a severe form of early-onset HH termed juvenile hemochromatosis (JH).2, 3 Mutations in HFE and TFR2 lead to adult-onset forms of HH.4, 5 Phenotypic analysis of patients with TFR2-associated HH (TFR2-HH) suggests that some may have earlier onset and more severe iron loading than patients with HFE-associated HH (HFE-HH).6–9 In patients with HFE-HH or TFR2-HH, hepcidin levels are decreased despite increased iron loading.10, 11 A similar disruption in hepcidin regulation has been observed in Hfe null mice10 and mice with mutated or deleted Tfr2.12, 13 Targeted deletion of Hfe or Tfr2 in hepatocytes recapitulates the iron overload phenotype typical of HH, and in both cases is associated with disrupted hepcidin regulation.14, 15 These studies suggest that Hfe and Tfr2 both function in hepatocytes and are required for the correct regulation of hepcidin in response to iron.

There has been one report of two siblings who displayed clinical symptoms characteristic of JH but did not carry mutations in either HAMP or HJV.16 However, they did show a combination of mutations in both the HFE and TFR2 genes.16 This observation suggests that HFE and TFR2 are complementary regulators of hepcidin expression. Although loss of either HFE or TFR2 results in an appreciable iron overload, with onset normally in adulthood, the loss of both would be predicted to lead to a combined loss of regulation, akin to that observed in JH.

To understand how Hfe and Tfr2 regulate hepcidin expression and, in turn, iron homeostasis we generated and characterized an Hfe and Tfr2 double-null mouse. We show that disruption of both Hfe and Tfr2 results in a more severe iron overload phenotype as compared to disruption of either gene individually. The severity of iron overload in the different strains of mice is related to the expression of hepcidin in relation to iron stores. Analysis of signaling pathways revealed that Hfe and Tfr2 may be working through the mitogen-activated protein kinase (MAPK) and mothers against decapentaplegic homolog (SMAD) pathways to regulate hepcidin expression.

Abbreviations

HAMP, hepcidin antimicrobial peptide; Hfe, hereditary hemochromatosis gene; HH, hereditary hemochromatosis; HIC, hepatic iron concentration; HJV, hemojuvelin; JH, juvenile hemochromatosis; Tfr2, transferrin receptor 2; TS, transferrin saturation.

Materials and Methods

Animals.

Animal studies were approved by the Queensland Institute of Medical Research Animal Ethics Committee. All animals had free access to water and food under standard conditions and received humane care. The Hfe−/− and Tfr2−/− mice have been described.10, 13 The original Hfe null mice were on a mixed C57BL/6 × SVJ/129 background (Professor William Sly, St. Louis University, USA). Mice were backcrossed to C57BL/6 for 10 generations. The two individual deletion strains were then crossed and the resultant double-heterozygous offspring used to produce all subsequent experimental mice. Maternal heterozygosity ensured that there were no adverse effects of maternal iron stores on the offspring during gestation and suckling. Mice were genotyped by polymerase chain reaction (PCR) analysis of tail DNA as described,17, 18 were weaned at 21 days, and maintained on standard laboratory chow ad libitum (iron content 120.1 mg/kg). After an overnight fast, male mice were sacrificed at 5 weeks of age and blood and tissues taken for further analysis.

Measurement of Iron Indices.

Transferrin saturation (TS) was measured using an iron and iron-binding capacity kit (Sigma-Aldrich, Castle Hill, Australia). Non-heme hepatic iron concentration (HIC) was measured using the method of Torrance and Bothwell.19 Iron was detected in the formalin-fixed liver sections using Perls' Prussian blue. Slides were viewed on an Olympus BX50 microscope and images captured using an Olympus DP11 digital camera (Olympus, Tokyo, Japan).

Real-Time PCR Analysis of Messenger RNA (mRNA) Transcripts.

Primer pairs for detecting iron-related mRNA transcripts are described (Supporting Table 1). Total RNA, isolated using Trizol reagent (Invitrogen, Mulgrave, Australia), was treated with RQ1 RNase free DNase (Promega, Annandale, Australia) before reverse transcription using Superscript III (Invitrogen). Real-time reaction mixes (20 μL) contained 5 μL of complementary DNA (cDNA) (diluted 1:10), 200 nM of each primer, and SYBR green PCR master mix (Applied Biosystems, Foster City, CA). Reactions, set up using the CAS1200 robot, were run on the Rotor-Gene 3000 (Corbett, Sydney, Australia). Expression of all target genes was normalized to β-actin levels.

Western Blotting.

Liver homogenates (100 μg) were electrophoresed on either 10% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or on 4%–12% Novex Bis-Tris gradient gels (Invitrogen) with a Tris or MES buffering system, respectively, transferred onto Hybond-C+ membrane, and blocked in 10% skim milk powder, 0.5% Tween 20 in TBS (Tris-buffered saline) (blocking buffer) overnight at 4°C. Anti-Tfr2 (0.1 μg/mL13), anti-Tfr1 (1 μg/mL; Invitrogen), anti-ferritin (1:2,000; Sigma-Aldrich), anti-prohepcidin (2.5 μg/mL20), anti-actin (1:1,000; Sigma-Aldrich), anti-phospho-Erk1/2 (1:1,000; Cell Signaling), anti-Erk1/2 (1:5,000; Cell Signaling), anti-phospho-Smad1/5/8 (1:1,000; Cell Signaling), and anti-Smad1 (1:250; Invitrogen) antibodies diluted in blocking buffer, or 5% bovine serum albumin (BSA) in 0.1% Tween 20 in TBS (TBS-T), were applied to the blots overnight at 4°C. Blots were washed with TBS-T and incubated with antirabbit or antimouse horseradish peroxidase (HRP) (1:10,000; Invitrogen) for 1 hour at room temperature. Following a final wash, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) was applied to the blots and exposed to film. Densitometry was performed using a SynGene Gene Genius Bioimaging System, bands were quantified using GeneTools (Syngene, Cambridge, UK), and levels were normalized to actin. Blots probed with phospho-specific antibodies were stripped by incubating with stripping buffer (1% SDS; 62.5 mM Tris-HCl, pH 6.8; 0.7% 2-mercaptoethanol) at 50°C for 30 minutes. Blots were then washed with TBS-T three times and reblocked with blocking buffer at room temperature for 1 hour.

Statistical Analysis.

Variables were compared between groups using both parametric (1-way analysis of variance [ANOVA] and Student's t test) and nonparametric (Kruskal-Wallis and Mann-Whitney) statistical tests using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Nonparametric tests were used when variances were significantly different. A P-value < 0.05 was considered statistically significant. Graphs were prepared using GraphPad Prism 4.0.

Results

Combined Deletion of Hfe and Tfr2 Results in Enhanced Iron Overload.

The proportion of offspring with the required genotype produced by the breeding pairs was not different to that expected for any of the three null strains (chi-squared test, Supporting Table 2). Five-week-old male mice (5-6 per group) were anesthetized and blood and tissues taken for analysis. The HIC and TS were significantly elevated in all null strains (Fig. 1A,B) with a gradient of increasing HIC and TS values from wildtype to Hfe−/− to Tfr2−/− to Hfe−/−/Tfr2−/− (Fig. 1A,B). HIC and TS were significantly elevated in the Hfe−/−/Tfr2−/− compared to Hfe−/− mice. The difference in TS between Hfe−/− and Tfr2−/− mice was also significant. Although the HIC and TS were greater in the Hfe−/−/Tfr2−/− than the Tfr2−/− mice, this did not reach statistical significance. Perls' staining of liver sections showed that all three null strains of mice had iron deposition in hepatocytes, with more intense staining observed in the Hfe−/−/Tfr2−/− livers reflecting the HIC (Fig. 1C). Iron loading in the Hfe−/− mice was predominantly periportal, expanding to include more pericentral regions in the Tfr2−/− and Hfe−/−/Tfr2−/− mice (Fig. 1C).

Figure 1.

Iron accumulation in Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice. (A) Hepatic iron concentration and (B) serum transferrin saturation were measured in wildtype (WT, n = 6), Hfe−/− (n = 5), Tfr2−/− (n = 5), and Hfe−/−/Tfr2−/− (n = 6) mice. Data are shown as box and whisker plots, showing median values, quartiles, and range. Statistical comparisons were performed using the Mann-Whitney test, significant difference (P < 0.01) compared to wildtype (a) and Hfe−/− (b). (C) Liver sections were stained with Perls' Prussian Blue for iron; 100× magnification images are shown.

Expression of Iron-Related mRNA and Protein Is Altered in Hfe/Tfr2 Double-Null Mice.

Real-time PCR was used to quantitate the expression of Hfe and Tfr2 mRNA in the mouse livers (Table 1). Hfe mRNA was absent in the livers of Hfe−/− and Hfe−/−/Tfr2−/− mice (Fig. 2A). There was a significant 1.5-fold elevation in Hfe mRNA (P < 0.001) in the Tfr2−/− mice compared to wildtype. Tfr2 mRNA was absent in the Tfr2−/− and Hfe−/−/Tfr2−/− mice (Fig. 2B). The absence of Tfr2 in Tfr2−/− and Hfe−/−/Tfr2−/− mice was confirmed by immunoblotting with anti-Tfr2 antibody (Fig. 3A,C). There was a significant 1.4-fold increase in the expression of Tfr2 protein (P < 0.001), but not mRNA in the livers of Hfe−/− mice compared to wildtype animals (Fig. 3C). This up-regulation of Tfr2 protein has been observed previously in Hfe−/− mice, and may be due to stabilization of Tfr2 in response to increased levels of diferric transferrin.21 The expression of iron-related mRNA transcripts was determined in the duodenum of all mice (Table 1). We found a significant increase in expression of the basolateral iron transporter ferroportin mRNA in Hfe−/−/Tfr2−/− mice (Table 1). Increases were also observed in the expression of the apical iron transporter Dmt1 and ferric reductase Dcytb mRNA in Tfr2−/− and Hfe−/−/Tfr2−/− duodenum, but this only reached statistical significance in the Hfe−/−/Tfr2−/− mice (Table 1). These results suggest that 5-week-old Hfe−/−/Tfr2−/− mice have up-regulation of components of the iron uptake machinery in the duodenum, consistent with increased iron absorption and the increased iron stores observed in these mice.

Table 1. Quantification of mRNA Transcripts in Liver and Duodenum of 5-Week-Old Wild-Type, Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− Mice
TissuemRNAWTHfe−/−Tfr2−/−Hfe−/−/Tfr2−/−
  • Values for each mRNA transcript are expressed as the mean relative to β-actin and normalized to wildtype (WT) values ± SD. Statistical significance compared to wildtype values are shown:

  • *

    P <0.05;

  • **

    P <0.01.

LiverHfe1.00 ± 0.180.01 ± 0.02**1.42 ± 0.25**0.01 ± 0.02**
 Tfr21.00 ± 0.681.08 ± 0.690.00 ± 0.00**0.02 ± 0.02**
 Tfr11.00 ± 0.471.10 ± 0.531.41 ± 0.390.72 ± 0.37
 Total Hamp1.00 ± 0.452.44 ± 0.71**0.93 ± 0.410.20 ± 0.25**
 Hamp11.00 ± 0.353.07 ± 1.07**1.35 ± 0.530.28 ± 0.39**
 Hamp21.00 ± 0.871.22 ± 0.380.11 ± 0.06**0.10 ± 0.10**
 Ferroportin1.00 ± 0.501.44 ± 0.571.09 ± 0.361.36 ± 0.49
 Dmt11.00 ± 0.561.03 ± 0.241.16 ± 0.231.19 ± 0.38
DuodenumTfr11.00 ± 0.291.55 ± 1.361.56 ± 0.651.06 ± 0.32
 Ferroportin1.00 ± 0.220.87 ± 0.341.16 ± 0.461.94 ± 1.25*
 Dmt11.00 ± 0.230.99 ± 1.035.30 ± 5.082.60 ± 0.85**
 Dcytb1.00 ± 0.340.96 ± 0.612.44 ± 1.692.62 ± 1.26**
Figure 2.

Hepatic mRNA expression in Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice. Real-time PCR was used to quantitate hepatic mRNA transcript levels in wildtype (WT, n = 6), Hfe−/− (n = 5), Tfr2−/− (n = 5), and Hfe−/−/Tfr2−/− (n = 6) mice. Results for hepatic expression of Hfe (A), Tfr2 (B), and Hamp1 (C) mRNA transcripts from Table 1 are shown. Data are shown as mean values ± standard error of the mean (SEM). Statistical comparisons were performed using the Mann-Whitney test, significant difference (P < 0.05) compared to wildtype (a), Hfe−/− (b), and Tfr2−/− (c).

Figure 3.

Hepatic protein expression in Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/−mice. (A) Total liver protein from wildtype (WT, n = 6), Hfe−/− (n = 5), Tfr2−/− (n = 5), and Hfe−/−/Tfr2−/− (n = 6) mice was immunoblotted with antibodies against Tfr1, Tfr2, prohepcidin, and ferritin; actin was used as a loading control. Protein levels were quantified by densitometry after normalizing to actin, (B) Tfr1, (C) Tfr2, (D) prohepcidin, (E) ferritin. Data are shown as mean values ± SEM. Statistical comparisons were performed using Student's t test or Mann-Whitney test, significant difference (P < 0.05) compared to wildtype (a), Hfe−/− (b) and Tfr2−/− (c).

Reduced Hepcidin Expression in Hfe/Tfr2 Double-Null Mice.

We have previously shown that both Hfe−/− and Tfr2−/− mice have suppressed hepatic hepcidin expression in relation to iron stores.10, 13, 15 These studies suggest that Hfe and Tfr2 play roles in signal transduction pathways regulating the expression of hepcidin. However, whether they function in the same or parallel pathways is unclear. To address this issue, we investigated hepcidin expression in Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice. The expression of Hamp1 mRNA was determined by quantitative real-time PCR (Fig. 2C). Surprisingly, Hamp1 mRNA was significantly elevated in the Hfe−/− mice. This result is considered in more detail in the next section. In Tfr2−/− mice the expression of Hamp1 was similar to wildtype. In the Hfe−/−/Tfr2−/− mice there was a significant decrease in the expression of Hamp1. The expression of the mouse homolog of Hamp1, Hamp2, and the combined expression of both Hamp subtypes (total Hamp) was also determined (Table 1). Hamp1 expression correlated closely with total Hamp, suggesting that Hamp1 is the predominant subtype expressed in the liver. Hamp2 expression was significantly decreased in Tfr2−/− and Hfe−/−/Tfr2−/− livers, but was unchanged in Hfe−/− livers compared to wildtype. Prohepcidin protein expression in the liver was also determined by Western blotting using prohepcidin antibody (Fig. 3A,D). The relative levels of prohepcidin protein correlated significantly with the expression of total Hamp and Hamp1 mRNA (R = 0.78 and R = 0.79, respectively, P < 0.001).

Hepcidin Expression Is Reduced in Relation to Iron Stores.

We have previously demonstrated a significant decrease in hepcidin expression in global and hepatocyte-specific Tfr2−/− mice at 5 weeks.15 The mice in the present study had a C57BL/6 background, whereas the mice in previous studies had a mixed 129T2/SvEms and C57BL/6 background.15 Various studies have shown that iron status and hepcidin expression can vary quite markedly between different mouse strains.22, 23 It has also been shown that hepcidin can vary with the age and gender of mice.15, 22, 24 Strain background may account for the high expression of hepcidin relative to controls observed in the Hfe−/− and Tfr2−/− mice when compared with previous studies.15 It has been suggested that although hepcidin expression is impaired in HFE-HH and in Hfe−/− mice, the liver is still capable of regulating hepcidin expression in relation to iron stores, albeit a blunted response.25–27 As there is a positive correlation between hepcidin levels and iron stores in both wildtype and Hfe−/− mice, we examined the expression of hepcidin relative to iron stores. We calculated hepcidin/ferritin and hepcidin/HIC ratios for all mice, using both Hamp mRNA and prohepcidin values. There was a decline in the Hamp1/ferritin ratio with a gradient from wildtype to Hfe−/− to Tfr2−/− to Hfe−/−/Tfr2−/− (Fig. 4A). The differences between each group were statistically significant, with the exception of the comparison between wildtype and Hfe−/− mice, which did not quite reach statistical significance (P = 0.0519, Mann-Whitney test). A similar pattern was observed for the Hamp1/HIC ratio (Fig. 4B). Differences in the prohepcidin/ferritin ratio between each group were also statistically significant (Fig. 4C), and prohepcidin/HIC ratios (Fig. 4D) followed the same trend.

Figure 4.

Hepatic hepcidin expression in relation to iron stores in Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/−mice. Ratios of Hamp1 mRNA relative to ferritin (A) and hepatic iron concentration (B), and prohepcidin relative to ferritin (C) and hepatic iron concentration (D) are shown for wildtype (WT, n = 6), Hfe−/− (n = 5), Tfr2−/− (n = 5), and Hfe−/−/Tfr2−/− (n = 6) mice. Data are shown as mean values ± SEM. Statistical comparisons were performed using the Mann-Whitney test, significant difference (P < 0.05) compared to wildtype (a), Hfe−/− (b), and Tfr2−/− (c).

Phosphorylation of Erk1/2 and Smad1/5/8 Is Altered in Null Mice.

We examined the potential signaling pathways through which Hfe and Tfr2 may be functioning to regulate hepcidin. The expression of the phosphorylated forms of three key signaling intermediates was determined by immunoblotting. Signal transducer and activator of transcription 3 (Stat3) phosphorylation is required for the up-regulation of hepcidin in response to the inflammatory cytokine IL-6.28 We observed no difference in the phosphorylation of Stat3 between the mouse strains (data not shown), suggesting that Stat3 is not involved in hepcidin regulation mediated by Hfe and Tfr2. HJV is a bone morphogenetic protein coreceptor and regulates hepcidin through the SMAD signaling pathway.29 We observed no significant difference in the levels of phospho-Smad1/5/8 in the Hfe−/− and Tfr2−/− livers (Fig. 5B). There was, however, a significant decrease in phospho-Smad1/5/8 in the Hfe−/−/Tfr2−/− livers relative to total Smad1. Similar decreases in Smad1/5/8 phosphorylation were observed in Hjv-null mice, and a link between Hjv and Smad signaling is well established.29 The relevance of the changes in Smad1 phosphorylation observed in the Hfe−/−/Tfr2−/− mice is unclear, but may suggest a role for Hfe and Tfr2 along with Hjv in the Smad signaling pathway regulating hepcidin. The MAPK signaling pathway has not been well studied in the regulation of iron homeostasis. One previous study using human cell lines suggested a role for the MAPK pathway in Tfr2 signaling.30 When we examined the phosphorylation of Erk1/2, we observed a significant decrease in the basal levels of phospho-Erk1/2 in the Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− livers when compared to wildtype (Fig. 5C). These results suggest that both Tfr2 and Hfe may be signaling through an Erk1/2-dependent pathway to regulate hepcidin.

Figure 5.

Basal levels of phosphorylated Smad1/5/8 and Erk1/2 in liver from Hfe−/−, Tfr2−/− and Hfe−/−/Tfr2−/−mice. Total liver protein from wildtype (WT), Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice was immunoblotted with antibodies against phospho-Smad1/5/8 and phospho-Erk1/2 (A). Blots were stripped and reprobed with antibodies against total Smad1 and Erk1/2; actin was used as a loading control. Protein levels were quantified by densitometry and results are expressed as ratios of phospho-protein/total protein/actin (white bars) and phospho-protein/total protein (black bars) for phospho-Smad1/5/8 (B) and phospho-Erk1/2 (C). All values are normalized to WT levels. Data are shown as mean values ± SEM. *P < 0.05; **P < 0.01.

Discussion

We generated an Hfe and Tfr2 double-null mouse to investigate the combined effects that deletion of these genes have on iron homeostasis. The backcrossing of mice onto a uniform genetic background has allowed, for the first time, a direct comparison of phenotype between the various null strains of mice. We have shown that Hfe−/−/Tfr2−/− mice develop more severe iron loading than either of the single null strains, and the Tfr2−/− mice have more severe iron loading than Hfe−/− mice. Iron accumulation in HH liver usually starts around the portal tracts, expanding to reach hepatocytes in all areas of the liver lobule in more advanced disease. Perls' staining in Hfe−/− mice had a distinctly periportal distribution, whereas the staining in Tfr2−/− and Hfe−/−/Tfr2−/− mice was consistent with more advanced disease. These observations are supported by several reports of patients with TFR2-HH with an earlier onset and more severe disease than patients with HFE-HH.6–9 The Hfe−/−/Tfr2−/− mouse described here is a model for the type of JH described in two siblings with mutations in both HFE and TFR2 genes.16 The severe iron loading in these patients results from a dramatic decrease in hepatic hepcidin expression in relation to iron stores.

The differences in iron loading phenotype between the strains of mice can be explained by variations in hepatic hepcidin expression. Surprisingly, we found a significant increase in hepcidin expression in 5-week-old Hfe−/− mice. This increase has not been observed in most previous studies of Hfe−/− mice and is likely due to strain background. The C57BL/6 strain used in this study has a “low iron” phenotype and higher hepcidin expression than other strains with a “high iron” phenotype.22 Pigeon et al.31 observed an increase in hepcidin mRNA in β2-microglobulin-deficient mice, compared to wildtype, suggesting that hepcidin can be elevated above wildtype levels in other animal models of HFE-HH. Several other studies suggest that although hepcidin expression is impaired in HFE-HH and in Hfe−/− mice, the liver is still capable of regulating hepcidin expression in relation to iron stores, albeit a blunted response.25–27. We corrected for iron stores by calculating hepcidin/ferritin and hepcidin/HIC ratios and showed that, although Hfe−/− mice had higher absolute hepcidin levels than wildtype mice, they actually have low hepcidin in relation to iron stores. Hepcidin expression in relation to iron stores was reduced further in Tfr2−/− mice and was lowest in the Hfe−/−/Tfr2−/− mice. These results suggest that with the loss of Hfe, hepatocytes are capable of regulating hepcidin expression in response to changes in iron load. Indeed, other studies have shown that hepcidin can be regulated in Hfe−/− mice and patients with HFE-HH, but basal expression levels are reduced, leading to inappropriately low hepcidin for a given iron load.25–27. It is likely that the regulation of hepcidin in Hfe−/− mice occurs through Tfr2; this is supported by the up-regulation of Tfr2 protein we observed in the Hfe−/− mice. Similarly, with the loss of Tfr2, hepcidin levels can be regulated to some extent, but this regulation is less efficient than that in wildtype and Hfe−/− mice. The up-regulation of Hfe mRNA in Tfr2−/− liver suggests that Hfe may be compensating for the loss of Tfr2, and is likely to be the source of the residual regulation of hepcidin in Tfr2−/− mice. With the loss of both Hfe and Tfr2, the regulation of hepcidin in response to iron is greatly reduced, indicating that both Hfe and Tfr2 are required for the correct regulation of hepcidin by iron. Taking the hepcidin expression results and phenotypic data together, we can conclude that Hfe and Tfr2 work in parallel (or possibly converging) signaling pathways, resulting in the regulation of hepcidin by iron. With the loss of one, the regulation of hepcidin can occur, albeit with reduced efficiency. With the loss of both, the regulation of hepcidin in response to iron is impaired further, resulting in more severe iron loading.

How HFE and TFR2 regulate hepcidin and the signaling pathways through which they act are unknown. An interaction between HFE and transferrin receptor 1 (TFR1) has been demonstrated,32 and a recent study suggested that HFE interacts with TFR2 in an iron-sensing complex.33 Another study showed that the HFE-TFR2 and HFE-TFR1 interactions are distinct, involving different protein domains and that HFE and Tf do not compete for binding sites on TFR2.34 A recent study characterized transgenic mouse strains with mutations in Tfr1 that either promoted a constitutive Hfe/Tfr1 interaction or interfered with Hfe/Tfr1 binding.35 They showed that with a constitutive Hfe/Tfr1 interaction, mice developed iron overload, associated with low hepcidin but when the interaction was interfered with mice developed iron deficiency, associated with high hepcidin.35 This suggests that when Hfe is free from Tfr1 it is able to initiate signaling to induce hepcidin transcription; this situation can occur when serum iron levels are increased and diferric-Tf displaces Hfe from its binding site on Tfr1. In the proposed model, the free Hfe molecule forms a hepcidin signaling complex with Tfr2, which is responsible for initiating signal transduction resulting in up-regulation of hepcidin transcription.35 This is supported by another recent study that concluded that liver cells require the interaction between HFE and TFR2 for holo-Tf induced hepcidin expression to occur.36 Our data comparing Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice are consistent with the findings of the Schmidt et al.35 study, that Hfe signals when free from Tfr1. However, our results suggest that the formation of a hepcidin signaling complex between Hfe and Tfr2 is not absolutely necessary to initiate hepcidin transcription in response to iron. If both Hfe and Tfr2 are required for signaling, then we would expect the phenotype of all three null strains to be identical. Rather, we see more severe iron loading and lower hepcidin in relation to iron stores in the Hfe−/−/Tfr2−/− mice, suggesting that Hfe and Tfr2 are still able to initiate signaling when not in complex with one another. We suggest that Hfe and Tfr2 are still able to signal in the absence of one another, but that the amplitude of the signal is greatly reduced, resulting in responsive but low basal hepcidin production. There has been only one study that linked Tfr2 with a potential signaling pathway. Utilizing the erythroleukemic cell line K562, Calzolari et al.30 suggested that diferric-Tf or anti-TFR2 antibodies could stimulate phosphorylation of p38-MAPK and ERK1/2. This study suggests that TFR2 could be working through a MAPK-dependent pathway to regulate hepcidin. Hjv, another important regulator of hepcidin, functions as a bone morphogenetic protein (BMP) coreceptor in hepatocytes and regulates hepcidin expression through the BMP/SMAD pathway.29 Recent studies have demonstrated the involvement of the Smad1/5/8 pathway in the regulation of hepcidin by iron.37, 38 It is possible that Hfe and Tfr2-dependent signaling pathways may converge with this pathway and affect Hjv-induced activation of hepcidin. Another recently published study has suggested that there may be crosstalk between the MAPK and the BMP/SMAD signaling pathways in the regulation of hepcidin by holotransferrin in hepatocytes.39 In this study they showed that both Erk1/2 and Smad1/5/8 are phosphorylated soon after addition of holotransferrin to mouse hepatocytes.39 The activation of Erk1/2 was attributed to the activation of Tfr2 by holotransferrin. Our analysis of phosphorylated Erk1/2 and Smad1/5/8 in the livers of mice also suggest that Erk1/2 or Smad1/5/8 are involved in the Hfe and Tfr2-dependent regulation of hepcidin by iron. A more detailed analysis of these pathways in mice or isolated hepatocytes challenged with iron or holotransferrin will be required to fully elucidate the role of Hfe, Tfr2, and the specific signaling pathways involved in hepcidin regulation. The phenotypic similarity between patients and animal models with Hjv-associated hemochromatosis and those with combined deletion or mutations in both Hfe and Tfr2, suggest that a common pathway involving Hfe, Tfr2, and Hjv may be involved in the regulation of hepcidin in response to iron. There is a complex interplay between signal transduction pathways and it is possible that other signaling pathways may be involved; the characterization of these signaling pathways will be important for our understanding of the pathogenesis of the various forms of hereditary hemochromatosis.

The phenotypic comparison of Hfe−/−, Tfr2−/− and Hfe−/−/Tfr2−/− mice on identical genetic backgrounds has demonstrated for the first time that loss of Tfr2 results in more severe iron loading than loss of Hfe. When extrapolated to humans our results suggest that TFR2-HH is a more severe iron loading disorder than HFE-HH. Iron loading is enhanced further by the loss of both Hfe and Tfr2, resulting in a more severe early onset hemochromatosis, similar in phenotype to JH caused by mutations in HJV or HAMP. Further research aimed at delineating the role of Hfe and Tfr2, the signaling pathways involved and the relationships between Hfe, Tfr2, and Hjv will be required to determine the roles of these molecules in regulating hepcidin and iron homeostasis.

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